Phosphorylation of native and heme-modified CYP3A4 by protein kinase C: A mass spectrometric characterization of the phosphorylated peptides

As an initial approach toward the characterization of the phosphorylation o f cumene hydroperoxide (CuOOH)-inactivated cytochrome P450 (CYP3A4, the maj or human liver drug-metabolizing enzyme) and its role in the degradation of the inactivated protein, we have identified one of the major participating cytosolic kinase(s) as rat liver cytosolic protein kinase C (PKC) with the use of specific and general kinase inhibitors. Accordingly, we employed a model phosphorylation system consisting of purified PKC, gamma -S-[P-32]ATP , and either native or CuOOH-inactivated purified recombinant His(6)-tagged CYP3A4. Lysylendoprotease (Lys)-C digestion of the phosphorylated CuOOH-in activated CYP3A4(His)(6) followed by HPLC-peptide mapping and mass spectrom etric (LC/MS/MS) analyses led to the isolation and the unambiguous identifi cation of two PKC-phosphorylated CYP3A4 peptides: E258SRLEDT(p)QK(266) and F414LPERFS(P)K-421. Similar analyses of the PKC-phosphorylated native enzym e predominantly yielded E258SRLEDT(p)QK(266) as the phosphorylated peptide. Studies are currently in progress to determine whether phosphorylation of any or both of these peptides is required for the Ub-dependent 26S proteaso mal degradation of CuOOH-inactivated CYP3A4.